In this day and age called the post-genomic era, instead of traditional researches based on heuristic approach, a new approach is required which is capable of developing biological macromolecules with various structures on the basis of comprehensive genome data, analyzing their molecular structures and functions, artificially designing a molecular structure based on the analytical result to prepare more highly functional pharmaceutical molecules and proteins, and applying them to drug discoveries, biological sciences, medical sciences or industries. The acquisition of a higher-quality macromolecule crystal is essential for performing the molecular structure analysis with a higher degree of accuracy, and thus an experimental test on crystallization of macromolecules is a very sort of basic and vital step. What is firstly required under these circumstances is to establish a technique for screening the conditions of a crystallization test in a simplified and speedy manner and preparing a high-quality crystal according to the obtained optimal crystallization conditions, and the establishment of such a technique is also a R & D that is societal demand. A primary technique for crystallization of macromolecules includes a vapor diffusion process, a batch process, a dialysis process and a liquid-liquid diffusion process.
Heretofore, the vapor diffusion process has been most widely used, because of relatively simple procedures of this process itself, and the existence of a large amount of information about crystallization accumulated in past years by many researchers and a number of commercially available products and screening kits using the vapor diffusion process.
However, optimal crystallization conditions for the vapor diffusion process can be found only if a crystallization test is repeatedly performed using a large number of combinations of various macromolecule samples and various precipitating-agent or precipitant solutions different in concentration. The reason comes from the fact that, in the search of crystallization conditions for the vapor diffusion process, the conditions of one sample drop can be searched only in the linear interval between a certain initial condition and a terminal condition determined by the concentration of a precipitant solution in a reservoir. In contrast, the crystallization process using liquid-liquid diffusion in a narrow tube or tubule allows almost all concentration conditions to be created at either point in a single tubule with time. Thus, it can be said that the liquid-liquid diffusion process in a tubule is simple and efficient as a technique for finding optimal crystallization conditions.
In the conventional liquid-liquid diffusion process, the crystallization of a macromolecule in a tubule has been performed by forming layers of a macromolecule sample and a precipitant solution in the tubule without mixing them up. In this technique, the macromolecule sample and the precipitant solution are diffused into one another through the interface therebetween. The precipitant solution generally has a diffusion rate greater than that of the macromolecule sample, and thereby the crystallization will be initiated in a region of the macromolecule sample.
Late years, in this technique, there has been developed a process of inducing the diffusion between the above two solutions through a gel layer having a function of buffering the crystal growth rate of a macromolecule. The use of this process can eliminate the need for taking account of preventing intermixing between the two solutions during formation of a double layer thereof. This process also has a feature in that the intervention of the gel allows the respective diffusion rates of the two solutions to be reduced so as to provide an extended diffusion time. It is desirable to induce the diffusion in sufficient time, because a macromolecule crystal prepared at a lower growth rate generally has a higher quality.
In the above conventional technique for forming a macromolecule crystal by means of liquid-liquid diffusion to be induced under the intervention of gel, a gel layer is formed in a vessel, and a tubule containing a sample of macromolecule is put in the vessel in such a manner as to insert the front end of the tubule into the gel layer by a given length. After the insertion, a precipitant solution is poured on the gel layer to form a double layer structure of the gel layer and the precipitant solution layer.
As time passes in this state, the precipitant solution in the precipitant layer is diffused into the gel layer, and then diffused from the front end of the tubule into the macromolecule sample solution in the tubule. In this way, an intended environment is created where the precipitant solution is brought into contact with the macromolecule sample within the tubule to form a macromolecule crystal. Then, as time further passes, the precipitant solution is continuously diffused to have a concentration gradient in which the concentration of the precipitant solution is gradually reduced in a direction from the front end toward the inside of the tubule. Concurrently, the macromolecule sample in the tubule is reversely diffused into the gel to have a concentration gradient in which the concentration of the macromolecule sample in the tubule is gradually reduced in a direction from the inside toward the front end of the tubule.
The presence of the above mutual concentration gradients allows an optimal point for crystal growth of the macromolecule to be provided with high probability so as to obtain an excellent crystal.
However, it has been pointed out that the above conventional crystal formation technique involves a problem about the risk of outflow of the macromolecule sample in the tubule, technical difficulties in inserting the tubule into the gel layer uniformly by a given length, and variation in sample setting operation in a case where it is required to prepare numbers and various types of samples, resulting in poor quality of an obtained crystal or poor process yield.
Further, the conventional technique cannot sufficiently meet the need for preparing a great number of macromolecule sample crystals in current researches.
Moreover, the device for the conventional technique involves a structural problem about ineffective consumption of precipitant and gel in large quantity.